Compositions for treating joints comprising bone morphogenetic protein and hyaluronic acid

ABSTRACT

Methods and compositions are disclosed for an intra-articular injection for the treatment of osteoarthritis. The methods and compositions comprising combinations of hyaluronic acid and a bone morphogenetic protein, like rhGDF-5, can be useful for any synovial joint, including the knee, shoulder, hip, ankle, hands, spinal facet, or temporomandibular joint, both for the relief of pain and for slowing disease progression.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.13/788,838, filed on Mar. 7, 2013, now U.S. Pat. No. 8,927,491, andentitled “METHODS FOR FORMING COMPOSITIONS FOR TREATING JOINTSCOMPRISING BONE MORPHOGENETIC PROTEIN AND HYALURONIC ACID,” which is adivisional of U.S. application Ser. No. 12/979,990, filed on Dec. 28,2010, now U.S. Pat. No. 8,455,436, and entitled “Compositions andMethods for Treating Joints,” each of which is incorporated by referenceherein in its entirety.

INCORPORATION OF SEQUENCE LISTING

The contents of the text file named “47062_030C01US_Sequence.txt,” whichwas created on Dec. 3, 2014 and is 20 KB in size, are herebyincorporated by reference in their entirety.

FIELD

The present invention relates generally to compositions and methods fortreating joints.

BACKGROUND

Osteoarthritis (“OA”), the most common form of arthritis, is a type ofarthritis that is characterized by degenerative (gradual deteriorationof joint) or abnormal changes in bone, cartilage, and synovium of thejoints. OA is often characterized by a progressive wearing down ofopposing joint surfaces accompanied at times by inflammation resultingin pain, swelling, and stiffness for the patient. OA can occur in one ormore joints following trauma to the joint, following an infection of thejoint, or simply as a result of aging. Furthermore, there is emergingevidence that abnormal anatomy may contribute to early development ofOA.

Treatment of OA generally involves a combination of exercise or physicaltherapy, lifestyle modification, and analgesics. Acetaminophen istypically the first line treatment for OA. For mild to moderatesymptoms, effectiveness is similar to non-steroidal anti-inflammatorydrugs (“NSAIDs”), such as ibuprofen. For more severe symptoms NSAIDs maybe more effective. However, while more effective, NSAIDs in severe casesare associated with greater side effects such as gastrointestinalbleeding and renal complications. Another class of NSAIDs, COX-2selective inhibitors (such as Celecoxib), are equally effective toNSAIDs but no safer in terms of side effects. There are several NSAIDsavailable for topical use, including diclofenac. Typically, they haveless systemic side-effects than oral administration and at least sometherapeutic effect. While opioid analgesics, such as morphine andfentanyl, improve pain this benefit is outweighed by frequent adverseevents and thus they are not routinely used. Intra-articular steroidinjections are also used in the treatment of OA, and they are veryeffective at providing pain relief. However, the durability of the painrelief is limited to 4-6 weeks and there are adverse effects that mayinclude collateral cartilage damage. If pain becomes debilitating, jointreplacement surgery may be used to improve mobility and quality of life.There is no proven treatment to slow or reverse the disease.

For patients who do not get adequate pain relief from simple painrelievers, like acetaminophen or from exercise and physical therapy,intra-articular injections of hyaluronic acid (HA) provide anothertreatment option to address symptomatic pain and delay the need for atotal joint replacement surgery. It is known that the concentration ofnative HA is deficient in individuals suffering from OA and thereforejoint injections of exogenous HA is believed to replenish thesemolecules and restore the viscoelastic properties of synovial fluid. Itis this property that is responsible for lubricating and cushioning thejoints. There is also evidence that HA has biological activity throughbinding to cell surface receptors and may have a role in mitigatinginflammation. Independent of the mechanism of action, pain relief isobserved for about six months following a treatment course. A treatmentcourse for HA products on the US market can range from single injectionproduct to others that require 3 to 5 weekly injections to attain thisdurability of pain relief.

There remains a need for improved methods and compositions for treatingOA joints, and to address the pain and structural degenerationassociated with OA.

SUMMARY

In one aspect, a method for treating a joint condition is disclosed. Themethod includes combining a solution of hyaluronic acid (HA) with a bonemorphogenetic protein (BMP) to form a mixture in which the BMP isprecipitated and dispersed in the solution of HA and the mixture has apH of at least about 3, preferably in the range of about 3 to 8, andmore preferably in the range of about 5-7.5. The method further includesinjecting the mixture into a subject to treat the OA joint condition,such that the BMP is capable of becoming solubilized and biologicallyactive within the subject after injection.

In one embodiment, the BMP can be a growth and differentiation factorprotein, such as growth and differentiation factor 5 (GDF-5), growth anddifferentiation factor 6 (GDF-6) and growth and differentiation factor 7(GDF-7). In another embodiment, the BMP can be BMP2 or BMP7.

In another embodiment, BMP is present in the mixture at a concentrationin the range of about 5 μg/ml to 2000 μg/ml, and more preferably in therange of about 5 μg/ml to 500 μg/ml. Furthermore, the BMP can be in aliquid state or a solid/lyophilized state which then can be combinedwith HA. When in a liquid form, the BMP can be solubilized in an acidsolution, e.g., hydrochloric acid, with a pH of less than about 4.

In yet another embodiment, the HA can have a molecular weight of atleast about 500 kilodaltons (kDa), and more preferably the HA has amolecular weight of at least about 1 million daltons. Additionally,compositions include HA at a concentration in the range of about 5 mg/mlto 60 mg/ml, and more preferably between 7 and 30 mg/ml. The HA can alsobe present in a liquid state or a solid/lyophilized state prior tocombining with the BMP. When in a liquid form, the HA can be solubilizedin water, saline or buffered solution, or any other diluents known inthe art. The HA solution can also have a pH in the range of about 5 to 9prior to combination. After combining the BMP with the HA solution, themixture can have a pH in the range of about 3 to 8.

In another aspect, a composition for treating a joint condition isdisclosed. The composition can be in the form of an injectableformulation that includes a precipitate of a bone morphogenetic protein(BMP) dispersed within a solution of hyaluronic acid (HA). Theinjectable formulation can have a pH of at least about 3. Moreover, theBMP is present in the injectable formulation in a precipitated form thatis capable of becoming solubilized and biologically active afterinjection into an organism.

In one more aspect, a method for forming a composition for treating ajoint disorder is disclosed. The method can include combining a solutionof hyaluronic acid (HA) and a bone morphogenetic protein (BMP) andallowing the combination to form a mixture containing a precipitate ofthe BMP that is dispersed within the HA solution. The BMP in theresulting composition is in its precipitated form and capable ofbecoming solubilized and biologically active following delivery to anorganism.

In yet another aspect, a kit is disclosed. The kit can include a firstcomponent being a solution of hyaluronic acid (HA) having a pH in therange of about 3 to 8, a second component including an amount of a bonemorphogenetic protein (BMP) that is in precipitate form and capable ofbecoming solubilized and biologically active following delivery to anorganism, and a syringe for injecting a mixture of the first componentand the second component. Moreover, the syringe can have a first chambercontaining the first component, a second container containing the secondcomponent and a plunger configured to inject the second component intothe first chamber to mix the first and second components.

BRIEF DESCRIPTION OF THE DRAWINGS

The appended drawings have been included herein so that theabove-recited features, advantages and objects will become clear and canbe understood in detail. These drawings form a part of thespecification. It is to be noted, however, that the appended drawingsillustrate exemplary embodiments and should not be considered to limitthe scope.

FIG. 1 is a perspective view of one embodiment of a mixing and deliverysystem for use with the compositions and methods of the presentinvention;

FIG. 2 is a perspective view of another embodiment of a mixing anddelivery system for use with the compositions and methods of the presentinvention;

FIG. 3 illustrates the method of preparation of the test solutions forELISA from a stock solution of rhGDF-5 in 10 mM HCl. Finalconcentrations of rhGDF-5 were made at 0.5, 0.05 and 0.005 mg/mL in therespective diluents;

FIG. 4 depicts standard ELISA techniques to assess homogeneity (at timet=0) and stability (at time t=0, 24, and 120 hrs) of batches of ˜2 mLpreparations with varying rhGDF-5 concentrations in 50 microliteraliquots;

FIG. 5 shows a graphical representation of formulations prepared andstored at 4° C. prior to ELISA testing to measure stability over time;

FIG. 6 illustrates the protocol for the bioactivity test in awell-established chondrogenic bioassay;

FIG. 7 illustrates graphical results of the chondrogenic assay tomeasure bioactivity; and

FIG. 8A is a photomicrograph illustrating in vivo and intra-articularbioactivity of rhGDF-5 in the HA formulations.

FIG. 8B is a photomicrograph illustrating untreated contralateral joint.

DETAILED DESCRIPTION

In general, the present invention provides compositions and methods fortreating joint conditions, such as pain associated with osteoarthritis.The compositions and methods utilize hyaluronic acid (“HA”) incombination with one or more Bone Morphogenetic Proteins (“BMP”). In oneembodiment the BMP is present as a precipitate that is dispersed withina solution of HA. The composition can be formulated as an injectableformulation that has a pH of at least about 3, and the pH can be in therange of about 3 to 8, more preferably 4 to 7.5, even more preferably 5to 7.5. The composition can be used in a method of treating a jointcondition by administering the composition to a subject, such as byinjection into the body of the subject (e.g., by injection into a joint)as discussed below. Various BMPs are suitable for use in thecomposition, as described below. Exemplary BMPs include a growth anddifferentiation factor (GDF), such as GDF-5, GDF-6, and GDF-7, and bonemorphogenetic proteins (BMPs), such as BMP2 and BMP7.

Hyaluronic Acid

Hyaluronic acid, HA, can have various formulations and can be providedat various concentrations and molecular weights. The terms “hyaluronicacid,” “hyaluronan,” and “HA” are used interchangeably herein to referto hyaluronic acids or salts of hyaluronic acid, such as the sodium,potassium, magnesium, and calcium salts, among others. These terms arealso intended to include not only elemental hyaluronic acid, buthyaluronic acid with other trace elements or in various compositionswith other elements. The terms “hyaluronic acid,” “hyaluronan,” and “HA”encompass chemical or polymeric or cross-linked derivatives of HA.Examples of chemical modifications which may be made to HA include anyreaction of an agent with the four reactive groups of HA, namely theacetamido, carboxyl, hydroxyl, and the reducing end. The HA used in thepresent application is intended to include natural formulations,synthetic formulations, or combinations thereof. The HA can be providedin liquid or solid formulations, and the HA can be in pure liquid formor in a solvent at various concentrations.

HA is a glycosaminoglycan (GAG), and in particular HA is an unbranchedpolysaccharide made up of alternating glucuronic acid and N-acetylglucosamine units. It is a viscoelastic material that that is also foundin the extracellular matrix of cartilage bound to collagen. Inparticular, HA is an important building component of aggregatedproteoglycans which impart resilient characteristics of articularcartilage. HA not only helps keep the cartilage that cushions jointsstrong and flexible, but it also helps increase supplies ofjoint-lubricating synovial fluid. HA abnormalities are a common threadin connective tissue disorders. HA can thus be used, to prevent, treat,or aid in the surgical repair of connective tissue disorders.

HA can be used in the compositions and methods of the present inventionat various molecular weights. Since HA is a polymeric molecule, the HAcomponent can exhibit a range of molecular weights, and almost anyaverage of modal molecular weight formulation of HA can be used in thecompositions and methods of the present invention, including LowMolecular Weight (“LWM”) Hyaluronan (about 500 to 700 kilodaltons (kDa),Medium Molecular Weight (“MMW”) Hyaluronan (700-1000 kDa), and HighMolecular Weight (“HMW”) Hyaluronan (1.0-4.0 million daltons (MDa)). Incertain exemplary embodiments, the HA has a molecular weight of at leastabout 500 kDa, and more preferably the HA is a High Molecular Weight(“HWM”) HA having a molecular weight of at least about 1 MDa. Themolecular weight can be, for example, 500, 600, 700, 800, 900, 1000,1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200,2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400,3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600,4700, 4800, 4900, 5000 kDa or more, or any range derivable therein. Itis expected that chemically modified HA's could have very differentmolecular weights than described above. A crosslinked HA can likewisehave much higher molecular weight than noted above. Regardless, thesematerials are also applicable in this invention.

Solvents that can be used to solubilize HA include, but are not limitedto, water, saline or other salt solutions, buffer solutions such asphosphate buffered saline, histidine, lactate, succinate, glycine, andglutamate, dextrose, glycerol, as well as combinations thereof.

A person skilled in the art will appreciate that the compositions andmethods of the present invention can include various other jointtreatment or excipients, including, for example, amino acids, proteins,saccharides, di-saccharides, poly-saccharides, nucleic acids, buffers,surfactants and mixtures thereof. Steroids, anti-inflammatory agents,non-steroidal anti-inflammatory agents, analgesics, cells, stabilizers,antibiotics, antimicrobial agents, anti-inflammatory agents, growthfactors, growth factor fragments, small-molecule wound healingstimulants, hormones, cytokines, peptides, antibodies, enzymes, isolatedcells, platelets, immunosuppressants, nucleic acids, analgesics, celltypes, viruses, virus particles, and combinations thereof.

The concentration of HA present in the mixture can also vary, but in anexemplary embodiment HA is provided at a pharmaceutically effectiveamount. In an exemplary embodiment, the HA has a concentration of atleast about 5 mg/ml, and more preferably at least about 7 mg/ml, andmore preferably at least about 10 mg/ml, and more preferably at leastabout 15 mg/ml, and in some embodiments the concentration can be atleast about 20 mg/ml. Suitable concentrations of HA include about 5mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 11 mg/mg, 12 mg/ml,13 mg/ml, 14 mg/ml, 15 mg/ml, 16 mg/ml, 17 mg/ml, 18 mg/ml, 19 mg/ml, 20mg/ml, 21 mg/ml, 22 mg/ml, 23 mg/ml, 24 mg/ml, 25 mg/ml, 26 mg/ml, 27mg/ml, 28 mg/ml, 29 mg/ml, 30 mg/ml, 31 mg/ml, 32 mg/ml, 33 mg/ml, 34mg/ml, 35 mg/ml, 36 mg/ml, 37 mg/ml, 38 mg/ml, 39 mg/ml, 40 mg/ml, 41mg/ml, 42 mg/ml, 43 mg/ml, 44 mg/ml, 45 mg/ml, 46 mg/ml, 47 mg/ml, 48mg/ml, 49 mg/ml, 50 mg/ml, 51 mg/ml, 52 mg/ml, 53 mg/ml, 54 mg/ml, 55mg/ml, 56 mg/ml, 57 mg/ml, 58 mg/ml, 59 mg/l, 60 mg/ml or more or anyrange derivable therein.

In one embodiment, the first component comprises an HA having a highmolecular weight (1 to 4 MDa) having a concentration in the range ofabout 7-40 mg/ml. One such product is Orthovisc® manufactured by AnikaTherapeutics, Inc. of Bedford, Mass. Orthovisc® is a sterile,non-pyrogenic, clear, viscoelastic solution of hyaluronan. Orthovisc®consists of high molecular weight (1.0-2.9 MDa), ultra-pure naturalhyaluronan dissolved in physiological saline and having a nominalconcentration of 15 mg/ml. Orthovisc® is isolated through bacterialfermentation. One skilled in the art will recognize that there arecompanies such as Shiseido and Lifecore who can produce high molecularweight HA through a bacterial fermentation process. Another example ofan HA product available in the United States with these characteristicsis Euflexxa®.

While HA alone can be effective to treat joint conditions, HA combinedwith additional agents should provide additional benefits. Inparticular, Bone Morphogenetic Proteins (BMPs) can help bone andcartilage regeneration by effecting chondrogenesis. This inventionaddresses the difficulty in administering HA in combination with BMPssince BMPs are not soluble at neutral pHs. BMPs are soluble in acidicsolutions, however, it is not desirable to have an HA formulation at asufficiently low pH to solubilize the BMP. This is because HA is notstable at low pH and will degrade with time. However, a product thatcombines BMP and HA and is maintained at a neutral/slightly acidic pHcan ensure that the BMP can remain stable during and after theprecipitation process, and demonstrate biological activity followingprecipitation and subsequent injection into a patient. Having a nearneutral pH formulation is also desirable from the perspective of thepatient because there could be discomfort to the patient who receivesinjections of such acidic solutions. Thus, it has been discovered thatthe use of solid BMPs dispersed in solution combined with HA providesubstantial benefits over HA alone. In particular, although the BMPsprecipitate when combined with HA at or near neutral pH, the BMPs areable to resolubilize and become biologically active after injection intoa patient's body. It is believed that the BMPs are not biologicallyactive (or they have reduced biological activity) in their solid,precipitated form in HA, however upon resolubilization after injectioninto a patient's body, the BMPs regain their biological activity and/orbecome more biologically active than in their solid, precipitated form.

Bone Morphogenetic Proteins

The term “bone morphogenetic proteins,” as used herein embraces theclass of proteins typified by representatives of the TGF-β familysubclass of true tissue morphogens. The BMPs that are useful caninclude, but are not limited to, growth and differentiation factors (inboth monomeric and dimeric forms) (such as GDF-5, GDF-6 and GDF-7) andbone morphogenetic proteins (such as BMP2 and BMP7).

All members of this family share common structural features, including acarboxy terminal active domain, and are approximately 97-106 amino acidsin mature length. They are translated as precursor proteins consistingof a prodomain, which is released proteolytically by members of thesubtilisin-like proprotein convertase family, which is important toactivate signaling that is conferred through the mature domain. Allmembers share a highly conserved pattern of cysteine residues thatcreate three intramolecular disulfide bonds and one intermoleculardisulfide bond. The active form can be either a disulfide-bondedhomodimer of a single family member or a heterodimer of two differentmembers. (See Massague Annu. Rev. Cell Biol. 6:957 (1990); Sampath, etal. J. Biol. Chem. 265:13198 (1990); Ozkaynak et al. EMBO J. 9:2085-93(1990); Wharton, et al. PNAS 88:9214-18 (1991); Celeste et al. PNAS87:9843-47 (1990); Lyons et al. PNAS 86:4554-58 (1989), U.S. Pat. No.5,011,691, and U.S. Pat. No. 5,266,683).

Osteogenic BMPs were initially identified by their ability to induceectopic endochondral bone formation. (See Cheng et al. “Osteogenicactivity of the fourteen types of human bone morphogenetic proteins” J.Bone Joint Surg. Am. 85A: 1544-52 (2003)). In particular, BMP2 (SEQ IDNO:1) and BMP7 (SEQ ID NO:2) play an important role in the developmentof bone and cartilage. BMP2 has been shown to stimulate the productionof bone. BMP7 also plays a key role in the transformation of mesenchymalcells into bone and cartilage.

Growth/differentiation factors (GDF-1 to GDF-15) are initiallysynthesized as larger precursor proteins which subsequently undergoproteolytic cleavage at a cluster of basic residues approximately110-140 amino acids from the C-terminus, thus releasing the C-terminalmature protein parts from the N-terminal prodomain. The maturepolypeptides are structurally related and contain a conserved bioactivedomain comprising six or seven canonical cysteine residues which isresponsible for the characteristic three-dimensional “cysteine-knot”motif of these proteins. The mature proteins contain seven conservedcysteine residues that are assembled into active secreted homodimers.GDF dimers are disulfide-linked with the exception of GDF-3 and GDF-9.It will be appreciated by one skilled in the art that the term “GDF” isused interchangeably with “rhGDF.”

GDF-5 (SEQ ID NO:3) is a morphogen which has been shown to promote cellproliferation, differentiation and/or tissue formation in severaltissues. The protein is also known as morphogenetic protein MP52, bonemorphogenetic protein BMP-14, and cartilage-derived morphogeneticprotein-1 (CDMP-1). GDF-5 is closely related to GDF-6 (SEQ ID NO:4) andGDF-7 (SEQ ID NO:5), all of which can be used according to the presentinvention in combination with HA. These three proteins form a distinctsubgroup of the TGF-β superfamily, thus displaying comparable biologicalproperties and an extraordinary high degree of amino acid sequenceidentity. It has repeatedly been demonstrated that members of theGDF-5/-6/-7 subgroup are primarily important inducers and regulators ofbone and cartilage as well as tendon/ligament.

Native GDF-5 proteins are homodimeric molecules and act mainly throughinteraction with specific receptor complexes which are composed of typeI and type II serine/threonine receptor kinases. The receptor kinasessubsequently activate SMAD proteins, which then propagate the signalsinto the nucleus to regulate target gene expression.

Biological molecules (biomolecules), such as BMPs, havethree-dimensional structure or conformation, and rely on this structurefor their biological activity and properties. Exposing thesebiomolecules to various environments such as variations in pH,temperature, solvents, osmolality, etc., can irreversibly change ordenature the conformational state of the biomolecule, rendering itbiologically inactive.

The chemistry and the three dimensional structure of each BMP familymember impacts the solubility of the protein in an aqueous environment.BMP-2 is readily soluble at concentrations greater than 1 mg/ml when thepH is below 6, and above pH 6 the solubility can be increased by theaddition of 1 M NaCl, 30% isopropanol, or 0.1 mM heparin (Ruppert, et alEur J Biochem 237, 295-302 (1996)). The solubility of BMP-7/OP-1 is alsolimited at neutral pH. The solubility of GDF-5 is much more limited thanthat of BMP-2 of -7, and GDF-5 is nearly insoluble in physiological pHranges and buffers. GDF-5 is only soluble in water at pH 2 to 4 (Honda,et al, Journal of Bioscience and Bioengineering 89(6), 582-589 (2000)).GDF-5 is soluble at an alkaline pH of about 9.5 to 12.0, howeverproteins degrade quickly under these conditions and thus acidicconditions have typically been used for preparation of GDF-5 protein.Solubility of these proteins are not only controlled by pH but are alsoaffected by the salt concentrations or other active ingredients in thesolution. For example, if there is an active ingredient in solution thatthe protein will bind to, it can cause insolubility of the protein.

Growth factors, such as BMPs, have been combined with other components.Due to the fact that solubility of most BMP's is limited at neutral pH,as noted above, BMP's are likely to precipitate out of solution inresponse to combination with neutral solutions, such as soluble HA. Manyefforts have been made to prevent the precipitation and/or increase themaintenance of a bioactive BMP when combined with such components.However, no combinations have been previously disclosed for theutilization of a solid form of BMP, either precipitate or lyophilized,in a HA solution. The present compositions and methods combine a solidor lyophilized formulation of BMP, such as rhGDF-5, with a liquidformulation of HA. In another embodiment, compositions and methods aredisclosed that form a mixture by combining a solution of BMP with asolution of HA, wherein the BMP forms a precipitate upon such acombination. These two components, solubilized HA and precipitated BMP,combined together form the mixture that is subsequently administered toa patient. As noted above, the three dimensional conformation is crucialfor the activity of proteins including growth factors. The act ofaggregating or precipitating such a molecule in solution can disturb thethree dimensional structure leading to loss of activity of the protein.Therefore, those skilled in the art have typically taken precautions toprevent change in conformation or structure of proteins because thesechanges can be irreversible.

One skilled in the art will appreciate that the term “precipitation,” asused herein, refers to the formation of an insoluble protein in thesolution. In contrast to known examples of drugs that are delivered as asuspension (due to the fact that the carrier, e.g., a mineral, ceramic,metal, or polymeric, is insoluble rather than the active protein) anaspect of the invention disclosed herein is the active precipitation ofthe protein immediately prior to delivery of the mixture to a patient.

When in a liquid formulation, the BMP can be provided in water, saline,an acid solution (e.g., hydrochloric acid, acetic acid, benzoic acid),another acidic solvent, or another solvent suitable for solubilizationof BMP.

The concentration of BMP present in the mixture can also vary, but in anexemplary embodiment BMP is provided at a pharmaceutically effectiveamount. In an exemplary embodiment, the BMP has a concentration of atleast about 0.1 μg/ml, and more preferably at least about 5 μg/ml, andmore preferably at least about 50 μg/ml, and more preferably at leastabout 200 μg/ml, and in some embodiments the concentration can be atleast about 500 μg/ml. Suitable concentrations of BMP include about 5μg/ml, 6 μg/ml, 7 μg/ml, 8 μg/ml, 9 μg/ml, 10 μg/ml, 11 μg/mg, 12 μg/ml,13 μg/ml, 14 μg/ml, 15 μg/ml, 16 μg/ml, 17 μg/ml, 18 μg/ml, 19 μg/ml, 20μg/ml, 21 μg/ml, 22 μg/ml, 23 μg/ml, 24 μg/ml, 25 μg/ml, 26 μmg/ml, 27μg/ml, 28 μg/ml, 29 μg/ml, 30 μg/ml, 31 μg/ml, 32 μg/ml, 33 μg/ml, 34μg/ml, 35 μg/ml, 36 μg/ml, 37 μg/ml, 38 μg/ml, 39 μg/ml, 40 μg/ml, 41μg/ml, 42 μmg/ml, 43 μg/ml, 44 μg/ml, 45 μg/ml, 46 μg/ml, 47 μg/ml, 48μg/ml, 49 μg/ml, 50 μg/ml, 60 μg/ml, 70 μg/ml, 80 μg/ml, 90 μg/ml, 100μg/ml, 150 μg/ml, 200 μg/ml, 250 μg/ml, 300 μg/ml, 350 μg/ml, 400 μg/ml,450 μg/ml, 500 μg/ml, 550 μg/ml, 600 μg/ml, 650 μg/ml, 700 μg/ml, 750μg/ml, 800 μg/ml, 850 μg/ml, 900 μg/ml, 950 μg/ml, 1000 μg/ml, 1500μg/ml, or 2000 μg/ml or more or any range derivable therein. A personskilled in the art can determine a suitable concentration of BMP frommethods known in the pharmaceutical arts, and that determination willgovern the nature and the concentration of BMP in the composition.

Lyophilization

Any one or more of the components present in the compositions andmethods of the present invention can be lyophilized using varioustechniques known in the art. Lyophilization is a dehydration processthat is typically used to preserve a perishable material, and it worksby freezing the material and then reducing the surrounding pressure andadding enough heat to allow the frozen water in the material to sublimedirectly from the solid phase to the gas phase. Standard lyophilizationtechniques known in the art can be used to lyophilize any one or more ofthe components. In an exemplary embodiment, at a minimum the one or moreBMPs are lyophilized.

Prior to lyophilization, various solvents can be used to form an aqueousmixture containing the component(s) to be lyophilized. In an exemplaryembodiment, the aqueous mixture is prepared by combining water with oneor more of the components. The component(s) can be present within themixture at various amounts, for example in the range of about 0.05 mg/mLto 10 mg/ml rhGDF-5. [there is no preferred]. In an exemplaryembodiment, the composition is filter sterilized, such as with a 0.2 μmfilter, prior to lyophilization.

In one embodiment, the component(s) can be lyophilized using thefollowing cycle:

Freezing: from ambient temperature to 5° C. in 15 minutes

-   -   Hold at 5° C. for 100 minutes    -   Down to −45° C. in 50 minutes    -   Hold at −45° C. for 180 minutes

Primary Drying: set pressure at 50 mTorr

-   -   Shelf Up to −15° C. in 175 minutes    -   Hold at −15° C. for 2300 minutes

Secondary Drying: set pressure at 75 mTorr

-   -   Shelf Up to 25° C. in 200 minutes    -   Hold for 900 minutes

Cycle end: backfill with nitrogen to ˜730 Torr

-   -   Capping and crimping

Variations to the temperatures, times and settings can be made inaccordance to practices used by a person of skilled in the art.Variations may include, but are not limited to, cycling temperatures forthe freezing cycle, drying temperatures and end cycles. Variations mayalso include differences in holding times for the freezing, drying andcapping/crimping cycles. Variations may also include differences in setpressures for the drying cycles and capping/crimping cycles. Inaddition, the number of drying cycles may be increased or decreaseddepending the machine used or component(s) to be lyophilized.

The addition of a buffering agent can provide for improved solubilityand stability of the protein in lyophilized formulations. Biocompatiblebuffering agents known in the art can be used, such as glycine; sodium,potassium, or calcium salts of acetate; sodium, potassium, or calciumsalts of citrate; sodium, potassium, or calcium salts of lactate; sodiumor potassium salts of phosphate, including mono-basic phosphate,di-basic phosphate, tri-basic phosphate and mixtures thereof. Thebuffering agents can additionally have sugar added to the composition tofunction as a bulking agent. The pH preferably can be controlled withinabout 2.0 to about 5.0 pH units, and more preferably within about 2.5 toabout 3.5 pH units.

Formulations

In an exemplary embodiment, the components are configured to be combinedintraoperatively, i.e., immediately before or during an operation. Thecomponents, when combined, can form a resulting composition or mixturehaving each component present in the composition at various amounts. Theamount of each component in the composition can vary, but in anexemplary embodiment, The mixing ratio between HA and BMP can have aweight ratio of HA to BMP in the range of about 1:0.001 to about 1:0.3,and more preferably at a ratio in the range of about 1:0.005 to about5:1. In other embodiments, a range of ratios, or more or any rangederivable therein, between about 1:0.005 to about 5:1 of HA to BMP canbe useful. Alternatively, compositions can include about 1% to about 75%or more by weight of each of the individual components, such as HA andBMP, in the total composition, alternatively about 2.5%, 5%, 10%, 20%,30%, 40%, 50%, 60%, 70% or more by weight of HA and BMP in the totalcomposition. In an exemplary embodiment, the amount of HA present in thedisclosed compositions is about 1-4% by weight of the total composition,and the amount of BMP present in the disclosed compositions is no morethan 2% by weight of the total composition.

Solvents that can be used to solubilize one or more of the componentsinclude, for example, water, acidic solvents, hydrochloric acid, aceticacid, benzoic acid, phosphate buffered saline, dextrose, glycerol,ethanol and the like, as well as combinations thereof. Solvents that canbe used to solubilize HA can include, but are not limited to, water,saline or other salt solutions, buffer solutions such as phosphatebuffered saline, histidine, lactate, succinate, glycine and glutamate,dextrose, glycerol, and other suitable solvents, as well as combinationsthereof. Solvents that can be used to solubilize the BMP can includewater, saline, hydrochloric acid, acetic acid, benzoic acid, acidicsolvent, and other solvents suitable for solubilization of BMPs. Thecompositions can also include other components, such as dispersionmedia, coatings, antibacterial and antifungal agents, isotonic andabsorption delaying agents, and the like that are physiologicallycompatible. Isotonic agents include, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition. Thecomposition can also include minor amounts of auxiliary substances suchas wetting or emulsifying agents, preservatives or buffers, whichenhance the shelf life or effectiveness of the composition.

The components and/or the resulting composition can be sterilized priorto use using various techniques known in the art. Sterile injectablemixtures can be prepared by incorporating the active compound(s) in atherapeutically effective or beneficial amount in an appropriate solventwith one or a combination of ingredients, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating a compound(s), such as HA or BMP, into a sterile vehiclewhich contains a basic dispersion medium and any required otheringredients. In the case of sterile powders for the preparation ofsterile injectable mixtures, some methods can include preparation ofvacuum dried and freeze-dried components which yield a powder of thecomposition plus any additional desired ingredients from a previouslysterile-filtered mixture thereof.

The compositions can be incorporated into pharmaceutical compositionssuitable for administration to a subject. Typically, the pharmaceuticalcomposition comprises HA and at least one BMP and a pharmaceuticallyacceptable carrier. As used herein, “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like that are physiologically compatible. Examples ofpharmaceutically acceptable carriers include one or more of water,hydrochloric acid, acetic acid, benzoic acid, acidic solvent, saline,phosphate buffered saline, dextrose, glycerol, ethanol and the like, aswell as combinations thereof. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition.Pharmaceutically acceptable carriers may further comprise minor amountsof auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the composition.

The compositions can be in a variety of forms. These include, forexample, liquid, semi-solid and solid dosage forms, such as liquidsolutions (e.g., injectable and infusible solutions), dispersions orsuspensions, tablets, pills, and powders. The preferred form depends onthe intended mode of administration and therapeutic application. Typicalpreferred compositions are in the form of injectable or infusiblesolutions, such as compositions similar to those used for in vivoinjection. The preferred mode of administration is parenteral (e.g.,intra-articular, subcutaneous, intraperitoneal, intramuscular). In oneembodiment, the composition can be administered by infusion or injectiondirectly into the target area, such as a joint. In another embodiment,the composition can be administered by intramuscular or subcutaneousinjection.

Sterile injectable solutions can be prepared by incorporating the activecompound in a therapeutically effective or beneficial amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

Delivery Systems

The methods and compositions encompass kits for treating articulardisorders, such as joints. The kits can comprise of a first component,such as HA, and a second component, such as at least one BMP. Bothcomponents can be housed in a single or separate chambers of a syringefor injecting a mixture of the first and second components. In oneembodiment, the BMP can be lyophilized GDF-5. In another embodiment, theBMP can be lyophilized GDF-6 or GDF-7. In another embodiment, the secondcomponent can comprise more than one lyophilized BMP, selected fromBMP2, BMP7, GDF-5, GDF-6 and GDF-7. In another exemplary embodiment, akit is provided having HA and BMP components. The HA component cancomprise about 2 ml of Orthovisc®, which contains about 30 mg ofhyaluronan, 18 mg of sodium chloride, and up to about 2.0 mL of waterfor injection. The HA has a molecular weight in the range of about 1.0to 4 MDa. The BMP component can comprise about 0.005 to 3 mg of solidBMP, supplied by, for example, and lyophilized using the protocoldiscussed above. The BMP component need not be lyophilized andalternatively can be in a solution, as noted above, before combinationwith the HA component.

Compounds can be stored separately to increase shelf-life. Theindividual compounds can be lyophilized or in solid form in onesyringe/cartridge with diluent or a second compound in a secondsyringe/cartridge. In one embodiment, one of the compounds is inlyophilized form or in solid form and the second compound is a solutioncapable of combining with the lyophilized/solid compound. An example canbe at least one lyophilized or solid BMP can be stored in a firstchamber and a solubilized HA can be stored in a second chamber. Inanother embodiment, both compounds can be lyophilized or in solid formand housed in a single or separate chambers of a syringe/cartridge. Inanother embodiment, compounds can be lyophilized directly in the syringeor cartridge.

Pre-filled dual-chamber syringes and/or cartridges can also be utilizedwith the components and compositions. Pre-filled dual-chamber syringesenable the sequential administration of two separate compositions with asingle syringe push, thereby replacing two syringes with one. Thebenefits of a single delivery capability include increasing the speedand ease of drug administration; reducing risk of infection by reducingthe number of connections; lowering the risk of drug administration orsequence errors, and quicker delivery of compositions requiringcombination prior to administration. The dual-chamber syringe canaccommodate lyophilized, powder or liquid formulations in the frontchamber combined with diluents, saline or buffer in the rear chamber.

Prefilled syringes can contain the exact deliverable dose of desiredcompounds and diluents. The prefilled syringes can contain volumes fromabout 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml,0.9 ml, 1.0 ml, 1.5 ml, 2 ml, 2.5 ml, 3 ml, 3.5 ml, 4 ml, 4.5 ml, 5 ml,5.5 ml, 6 ml, 6.5 ml, 7 ml, 7.5 ml, 8 ml, 8.5 ml, 9 ml, 9.5 ml, 10 ml ormore or any derivative therein.

The dual syringe and/or cartridge can be side-by-side chambers withseparate syringe plungers that mix into a single chamber or linearchambers with one plunger. The dual chamber syringe and/or cartridgescan also have a stopper or connector in the middle to serve as a barrierbetween the two chambers. The stopper or connector can be removed toallow mixing or combining of the compounds in the two chambers.

FIG. 1 illustrates one embodiment of a mixing and delivery system thatis in the form of a dual chamber syringe 10. As shown, the dual chambersyringe 10 generally includes a housing having proximal and distalchambers 14, 12 separated by a valve 16. A plunger 18 is slidablydisposed within the proximal chamber 14 and is configured to injectfluid present within the proximal chamber 14 into the distal chamber 12to thereby mix the components. In one embodiment, the first component,e.g., liquid HA, can be present in the proximal chamber 14 and thesecond component, e.g., one or more BMPs, can be present in the distalchamber 12. The plunger 18 can be advanced through the proximal chamber14 to inject the first component, e.g., liquid HA, into the distalchamber 12 containing the second component, e.g., one or more BMPs. Inanother embodiment, the proximal chamber 14 can contain a solvent, suchas water or saline, and the distal chamber 12 can contain all of thecomponents in solid form. For example, the distal chamber 12 can containlyophilized or solid HA and one or more BMPs. The plunger 18 can beadvanced through the proximal chamber 14 to inject the solvent into thedistal chamber 12, thereby solubilizing the components in the distalchamber 12. Once all components are combined in the distal chamber 12,the mixture can be delivered to tissue, for example by attaching aneedle to the distal end of the dual chamber syringe.

FIG. 2 illustrates another embodiment of a mixing and delivery system20, which is sold commercially under the trade name MixJect®. In thisembodiment, the system includes a fluid control assembly 22 that iscoupled between a syringe 24 and a vial 26. The syringe 24 defines afirst chamber 24 a (not labeled in figure) which can contain a liquid,such as liquid HA or a solvent, and the vial defines a second chamber 26a (not labeled on figure) which can contain a solid, such as one or moreBMPs. Deployment of the plunger 28 through the syringe 24 will injectthe liquid through the control system and into the vial 26, where thesolid will be solubilized by the liquid. Once the components are fullysolubilized, the vial 26 can be inverted and the plunger 28 can beretracted to draw the mixture back into the chamber 24 a in the syringe24. The vial 26 can then be removed from the system, and the mixture canbe injected from the syringe through a needle 29 and into tissue.

A person skilled in the art will appreciate that any dual chambersystems known in the art can be used, and that the chambers can beside-by-side chambers with separate syringe plungers that mix into asingle chamber or linear chambers with a single plunger.

Treatments

The method and compositions can be administered, for in vivoapplications, parenterally by injection or by gradual perfusion overtime. Administration may be intraarticular, intravenous,intraperitoneal, intramuscular, subcutaneous, intracavity, ortransdermal. For in vitro studies the agents may be added or dissolvedin an appropriate biologically acceptable buffer and added to a cell ortissue.

The HA and inactive BMP can be co-administered or simultaneouslyadministered in the same formulation or in two different formulationsthat are combined via the same route. The HA and BMP components can becombined just prior to administration of the HA and inactive BMP. Thecombination can occur within seconds, minutes, hours, days or weeksprior to the administration of the composition.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's intravenousvehicles include fluid and nutrient replenishers, electrolytereplenishers (such as those based on Ringer's dextrose), and the like.Preservatives and other additives may also be present such as, forexample, anti-microbials, anti-oxidants, chelating agents, growthfactors and inert gases and the like.

Frequently used “carriers” or “auxiliaries” include magnesium carbonate,titanium dioxide, lactose, mannitol and other sugars, talc, milkprotein, gelatin, starch, vitamins, cellulose and its derivatives,animal and vegetable oils, polyethylene glycols and solvents, such assterile water, alcohols, glycerol and polyhydric alcohols. Intravenousvehicles include fluid and nutrient replenishers. Preservatives includeantimicrobial, anti-oxidants, chelating agents and inert gases. Otherpharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike, as described, for instance, in Remington's PharmaceuticalSciences, 15th ed. Easton: Mack Publishing Co.: 1405-1412, 1461-1487,1975 and The National Formulary XIV., 14th ed. Washington: AmericanPharmaceutical Association, 1975 the contents of which are herebyincorporated by reference. The pH and exact concentration of the variouscomponents of the pharmaceutical composition are adjusted according toroutine skills in the art. See Goodman and Gilman's The PharmacologicalBasis for Therapeutics (7th ed.).

Examples of symptoms or diseases, for which the composition and methodsdisclosed herein can be useful, encompass treating articular disorders,such as arthritis caused by infections, injuries, allergies, metabolicdisorders, etc., rheumatoids such as chronic rheumatoid arthritis, andsystemic lupus erythematosus; articular disorders accompanied by gout,arthropathy such as osteoarthritis, internal derangement, hydrarthrosis,stiff neck, lumbago, etc. Varying the effects depending on the use ofthe composition or the types of diseases to be treated, the agent canexert desired prophylactic and alleviative effects, or even therapeuticeffects on swelling, pain, inflammation, and destroying of articulationswithout seriously affecting living bodies. The composition for treatingarticular disorder can be used to prevent the onset of articulationdisorders, as well as to improve, alleviate, and cure the symptoms aftertheir onsets.

The methods of treatment can include directly injecting the compositionsinto the target area, such as a joint. Injections can be performed asoften as daily, weekly, several times a week, bi monthly, several timesa month, monthly, or as often as needed as to provide relief ofsymptoms. For intra-articular use, from about 1 to about 40 mg/ml of HAand one or more BMPs per joint, depending on the size of the joint andseverity of the condition, can be injected. The frequency of subsequentinjections into a given joint are spaced to the time of recurrence ofsymptoms in the joint. Illustratively, dosage levels in humans of thecomposition can be: knee, about 0.001 to about 40 mg/ml per jointinjection; shoulder, about 0.001 to about 40 mg/ml of HA and one or moreBMPs per joint injection; metacorpal or proximal intraphalangeal, about0.001/ml to about 40 mg/ml of HA and one or more BMPs per jointinjection; and elbow, about 1 to about 300 mg per joint injection.

It will be understood, however, that the specific dosage level for anyparticular patient will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination and the severity ofthe particular disease undergoing therapy. The pharmaceuticalcompositions can be prepared and administered in dose units. Undercertain circumstances, however, higher or lower dose units may beappropriate. The administration of the dose unit can be carried out bothby single administration of the composition or administration can beperformed in several smaller dose units and also by multipleadministrations of subdivided doses at specific intervals.

In one embodiment, the medical condition is osteoarthritis (OA) and thecomposition is administered in a joint space, such as, for example, aknee, shoulder, temporo-mandibular and carpo-metacarpal joints, elbow,hip, wrist, ankle, and lumbar zygapophysial (facet) joints in the spine.The viscosupplementation may be accomplished via a single injection ormultiple intraarticular injections administered over a period of weeksinto the knee or other afflicted joints. For example, a human subjectwith knee OA may receive one, two, or three injections of about 2, 3, 4,5, 6, 7, 8, 9, 10 ml or more per knee. For other joints, theadministered volume can be adjusted based on the size on the joint.

It will be understood, however, that the specific dosage level for anyparticular patient will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination and the severity ofthe particular disease undergoing therapy.

EXPERIMENTAL DATA Example 1 In Vitro Evaluations

Multiple in vitro studies were performed to test the hypothesis that theobserved rhGDF-5 precipitation in HA would negatively impact thebioactivity/potency of rhGDF-5.

Combinations of rhGDF-5 in different diluents with and without HA werecompared for the extent of rhGDF-5 precipitation. rhGDF-5 was suspendedin 10 mM HCl pH 2.0, which was then combined with (1) phosphate-bufferedsaline (PBS), (2) 1 mM HCl, (3) 0.9% saline, and (4) trehalose plusexcipients (F-18) to form separate formulations. Each of theseformulations was subsequently mixed with HA (Orthovisc®) with a finalconcentration of rhGDF-5 of 0.5 mg/mL in 0.5% HA. HA controlformulations were made by combining the respective diluents(phosphate-buffered saline (PBS), 1 mM HCl, 0.9% saline and Trehaloseplus excipients) with HA in the absence of rhGDF-5 to a finalconcentration of 0.5% HA, and HA-free controls were made by furtherdiluting the intermediate rGDF-5 formulations in diluent to a finalconcentration of 0.5 mg/mL rhGDF-5. The pH of the resultant formulationswas determined and they were visually assessed for rhGDF-5precipitation.

Precipitation of rhGDF-5 was observed in the presence of HA formulationsand PBS alone. Different aliquots of the same formulations demonstratedminimal variation. The extent of rhGDF-5 precipitation correlated withrhGDF-5 concentration.

As detailed below in Table 1, relative to 0.5% HA control formulations,the combination of rhGDF-5 at 0.5 mg/ml with HA resulted in cloudyprecipitants for all formulations. Select formulations were furtherevaluated, including rhGDF in PBS or PBS+HA and rhGDF-5 in F18(trehalose+excipients) or F18+HA. rhGDF-5 in 1 mM HCl (pH 3.0) alone wasincluded as a positive control as the protein is known to remain fullysoluble at this pH.

TABLE 1 Visual assessment for rhGDF-5 precipitation and pH GroupFormulation pH rhGDF-5 Solubility 1 PBS 6.8 No, Cloudy 2 PBS + HA 6.6No, Cloudy 3 F18 4.9 No, Particulates 4 F18 + HA 4.9 No, Slightly Cloudy5 HCl (1 mM) 2.7 Yes, Clear 6 HCl + HA 3.9 No, Cloudy + Aggregates 7Saline (0.9%) 2.9 Yes, Clear 8 Saline + HA 3.9 No, Cloudy + Aggregates *Formulations prepared using rhGDF-5 Batch in 10 mM HCl, pH = 2.0 *** PBSpH = 7.1, F18 pH = 5.4, 1 mM HCl pH = 3.0, 0.9% Saline pH = 5.1, HA pH =5.7

Formulation Homogeneity

FIG. 3 details the method used for preparation of test solutions forELISA from a stock solution of rhGDF-5 in 10 mM HCl. Batches of ˜2 mLwere prepared with final concentrations of rhGDF-5 at 0.5, 0.05 and0.005 mg/mL in the respective diluents. rhGDF-5 concentrations weredetermined in 50 microliter aliquots using standard ELISA techniques toassess homogeneity (at time t=0) and stability (at time t=0, 24, and 120hrs) of the resulting formulation.

FIG. 4 shows the correlation to the theoretical solution concentrationthat was injected, even though rhGDF-5 was observed to have precipitatedin the presence of HA formulations and PBS alone. Additionally, theconcentrations demonstrated minimal variation within the differentaliquots of the same formulation. Surprisingly, homogeneity ofprecipitate rhGDF-5 in the HA formulations was observed without regardto the relative viscosity of the HA solution.

Formulation Stability

The stabilization of the formulations was also tested by ELISA.Formulations were prepared at time t=0 and stored at 4° C. prior toELISA testing. FIG. 5 shows that stability of rhGDF-5 by HA in anaqueous solution near neutral pH (˜6.6) was observed, even over shorttime frames. Relative to the formulation of rhGDF-5 in PBS alone,rhGDF-5 in PBS+HA demonstrated less variation in solution concentrationand improved stability over time, as rhGDF-5 concentrations werecomparable to the theoretical solution concentration even out to 120hours at all doses/concentrations evaluated. Therefore, it is believedthat HA imparts stability to the protein especially at lowconcentrations (e.g., less than 2.5 ug) because the ELISA data (see FIG.5) show that after 24 and 120 hours, more protein is recovered than whenthe protein is in PBS alone. This implies that the presence of HA isimportant for preserving the activity of the growth factor. That is, tothe extent that the protein is biologically inactive in its solid,precipitated form, it becomes biologically active after injection into apatient. Similar results were observed for rhGDF-5 formulated intrehalose+excipients (F18) with and without HA, as solutionconcentrations appeared to remain stable over the time frame evaluated.The pH of the F18 and F18+HA solutions was ˜4.9. As expected, the HClonly control group (pH=2.7) was stable over time at each of theconcentrations evaluated.

Bioactivity

Bioactivity of the formulations was also assessed in a well-establishedchondrogenic bioassay assessing dose-dependent sulfatedglycosaminoglycan (sGAG) concentration produced by pellet cultures ofjuvenile bovine chondrocytes, which is shown in FIG. 6. The methodinvolved pelleting ˜250,000 chondrocytes and culturing them in presenceof a serum-free media combined with rhGDF-5 formulations in HA or apositive control formulation of rhGDF-5 in 1.0 mM HCl. Cultures werethen maintained for 14 days with media changes containing fresh rhGDF-5formulations every 3-4 days. At the experimental termination, cultureswere digested in papain and assayed for sGAG accumulation using a wellestablished DMMB (dimethylmethylene blue) absorbance assay. Testarticles were prepared as detailed in the schematic of FIG. 6 (theconcentrations denote the working concentration of rhGDF-5).

FIG. 7 summarizes the results from the chondrogenic assay. It wasobserved that the rhGDF-5 formulations containing HA demonstrated acomparable dose-dependent bioactivity to “naked” rhGDF-5 protein in 1 mMHCl despite the observation that protein was precipitated in the HAformulations. This demonstrates that the suspension of rhGDF-5 in HA isactive after injection into a patient.

Example 2 In Vivo Evaluations

An in vivo animal study was conducted to evaluate the intra-articularactivity of rhGDF-5 formulated in HA. Intra-articular injections ofrhGDF-5/HA formulations, shown in Table 2, were evaluated innormal/healthy rat knees to assess tolerance to the injection andintra-articular bioactive response. Semi-weekly injections wereperformed for a total of 3 weeks (6 injections total) of rhGDF-5formulated in PBS+0.5% HA (Orthovise), and a total of 7 animals pergroup were evaluated. The study design is detailed in Table 2.

TABLE 2 Intra-articular injection of rhGDF-5/HA formulations InjectionSolution Dose Solution Total Dose Group Name (ug/ml) (ug/ml) (ug/ml)rhGDF-5 Low 5 0.25 1.5 rhGDF-5 Middle 50 2.5 15 rhGDF-5 High 500 25 150

Based on the study design to evaluate a two-decade dilution series ofrhGDF-5, and HA only control was not included in this screening study.The results of the study demonstrated the injection series waswell-tolerated in all study groups as no lameness or inflammation wasobserved. An rhGDF-5 dose-dependent response was observed in the studygroups, as the high dose group (25 micrograms per injection)demonstrated the most pronounced intra-articular effects (see FIG. 8A)as opposed to the untreated contralateral joint (see FIG. 8B). Mildsynovitis and chondrogenesis was observed in the marginal zones of thehighest dose (25 micrograms per injection) study group. In vivo andintra-articular bioactivity was observed regardless of precipitation ofthe rhGDF-5 in the HA formulations.

TERMINOLOGY

A therapeutically effective amount or effective amount can beadministered of the composition to achieve a pharmacological effect. Theterm “therapeutically effective amount” includes, for example, aprophylactically effective amount. An “effective amount” is an amounteffective to achieve a desired pharmacologic effect or therapeuticimprovement without undue adverse side effects. For example, aneffective amount refers to an amount that increases operativity, orincreases weight bearing load, or decreases pain, or increases growth inthe bone and cartilage of one or more joints, or reduces jointdistortion, pain, swelling, or stiffness. The effective amount of anagent will be selected by those skilled in the art depending on theparticular patient and the disease level. It is understood that “aneffect amount” or “a therapeutically effective amount” can vary fromsubject to subject, due to variation in metabolism of therapeutic agentssuch as s and/or prokinetic agents, age, weight, general condition ofthe subject, the condition being treated, the severity of the conditionbeing treated, and the judgment of the prescribing physician.

“Treat” or “treatment” refers to any treatment of a disorder or diseaseassociated with bone or cartilage disorder, such as preventing thedisorder or disease from occurring in a subject which may be predisposedto the disorder or disease, but has not yet been diagnosed as having thedisorder or disease; inhibiting the disorder or disease, e.g., arrestingthe development of the disorder or disease, relieving the disorder ordisease, causing regression of the disorder or disease, relieving acondition caused by the disease or disorder, or stopping the symptoms ofthe disease or disorder. Thus, as used herein, the term “treat” is usedsynonymously with the term “prevent.”

By “co-administered” is meant simultaneous administration in the sameformulation or in two different formulations that are combined into oneformulation for administration. In one embodiment, the HA and the BMPare co-administered via delivery in the same formulation.

The term “subject” as used herein refers to an animal, preferably amammal and more preferably human who can benefit from the compositionsand methods of the present invention. There is no limitation on the typeof animal that could benefit from the present methods. A subjectregardless of whether a human or non-human animal may be referred to asan individual, subject, animal, host or recipient. The methods of thepresent invention have applications in human medicine, veterinarymedicine as well as in general, domestic or wild animal husbandry.Preferably, the candidate subject is a mammal such as a human orlaboratory test animal such as a mouse, rat, rabbit, guinea pig, hamsteror avian species such as a poultry bird.

One skilled in the art will appreciate further features and advantagesof the invention based on the above-described embodiments. Accordingly,the invention is not to be limited by what has been particularly shownand described, except as indicated by the appended claims. Allpublications and references cited herein are expressly incorporatedherein by reference in their entirety.

What is claimed is:
 1. A composition for treating a joint condition,comprising: an injectable formulation comprising a precipitate of a bonemorphogenetic protein (BMP) dispersed within a solution of hyaluronicacid (HA), the injectable formulation having a concentration of BMP inthe range of about 5 μg/ml to about 90 μg/ml and a concentration of HAin the range of about 5 mg/ml to about 60 mg/ml, and the BMP being inits precipitated form and capable of becoming solubilized andbiologically active after injection into an organism.
 2. The compositionof claim 1, wherein the BMP is a growth and differentiation factor (GDF)protein.
 3. The composition of claim 2, wherein the GDF is growth anddifferentiation factor 5 (GDF-5), growth and differentiation factor 6(GDF-6), growth and differentiation factor 7 (GDF-7), or a combinationthereof.
 4. The composition of claim 1, wherein the BMP is in a liquidstate prior to combining with the solution of HA.
 5. The composition ofclaim 1, wherein the BMP is in a lyophilized state prior to combiningwith the solution of HA.
 6. The composition of claim 1, wherein the BMPis at least one of BMP2 and BMP7.
 7. The composition of claim 1, whereinthe mixture has a pH in the range of about 3 to
 8. 8. The composition ofclaim 1, wherein the mixture has a pH in the range of about 5 to 7.5. 9.The composition of claim 1, wherein the HA has a molecular weight of atleast about 500 kDa.
 10. The composition of claim 1, wherein thesolution of HA comprises HA and water.
 11. The composition of claim 1,wherein the HA is at a concentration of at least 7 mg/ml in the mixture.12. A kit, comprising: a first component comprising a solution ofhyaluronic acid (HA); a second component comprising an amount of a bonemorphogenetic protein (BMP), the BMP configured to form a precipitatewhen mixed with the HA and capable of becoming solubilized andbiologically active after injection into an organism; and a syringe forinjecting a mixture of the first component and the second component,wherein the mixture has a concentration of BMP in the range of about 5μg/ml to about 90 μg/ml and a concentration of HA in the range of about5 mg/ml to about 60 mg/ml.
 13. The kit of claim 12, wherein the syringehas a first chamber containing the first component, and a secondcontainer containing the second component, and wherein the syringeincludes a plunger configured to inject the second component into thefirst chamber to mix the first component and the second component. 14.The kit of claim 12, wherein the BMP is at least one of BMP2 and BMP7.15. The kit of claim 12, wherein the BMP is a growth and differentiationfactor (GDF) protein.
 16. The kit of claim 15, wherein the GDF is growthand differentiation factor 5 (GDF-5), growth and differentiation factor6 (GDF-6), growth and differentiation factor 7 (GDF-7), or a combinationthereof.
 17. The kit of claim 12, wherein the mixture has a pH in therange of about 5 to 7.5.
 18. The kit of claim 12, wherein the HA has amolecular weight of at least about 500 kDa.
 19. The kit of claim 12,wherein the HA is at a concentration of at least 7 mg/ml in the mixture.